The present invention relates to the identification of certain human genes as cellular targets for the design of therapeutic agents for suppressing human immunodeficiency virus (HIV) infection. These genes encode products that are necessary for HIV infection, because HIV infection is inhibited when expression of these genes is down-regulated. Therefore, compounds that inhibit expression of these genes or inhibit function of the encoded gene products can be used as therapeutic agents for the treatment and/or prevention of HIV infection. In addition, the invention relates to methods for identifying additional cellular genes as therapeutic targets for suppressing HIV infection, and methods of using such cellular genes and their encoded products in screening assays for selecting protective compounds that inhibit HIV infection. The present invention also includes a method to prevent tumorigenesis.
The primary cause of acquired immunodeficiency syndrome (AIDS) has been shown to be HIV (Barre-Sinoussi et al., 1983, Science 220:868-870; Gallo et al., 1984, Science 224:500-503). HIV causes immunodeficiency in an individual by infecting important cell types of the immune system, which results in their depletion. This, in turn, leads to opportunistic infections, neoplastic growth and death.
HIV is a member of the lentivirus family of retroviruses (Teich et al., 1984, RNA Tumor Viruses, Weiss et al., eds., CSH-Press, pp. 949-956). Retroviruses are small enveloped viruses that contain a diploid, single-stranded RNA genome, and replicate via a DNA intermediate produced by a virally-encoded reverse transcriptase, an RNA-dependant DNA polymerase (Varmus, 1988, Science 240:1427-1439). There are at least two distinct subtypes of HIV: HIV-1 (Barre-Sinoussi et al., ibid.; Gallo et al., ibid.) and HIV-2 (Clavel et al., 1986, Science 233:343-346; Guyader et al., 1987, Nature 326:662-669). Genetic heterogeneity exists within each of these HIV subtypes.
CD4+ T cells are the major targets of HIV infection because the CD4 cell surface protein acts as a cellular receptor for HIV attachment (Dalgleish et al., 1984, Nature 312:763-767; Klatzmann et al., 1984, Nature 312:767-768; Maddon et al., 1986, Cell 47:333-348). Viral entry into cells is dependent upon viral protein gp120 binding to the cellular CD4 receptor molecule (McDougal et al., 1986, Science 231:382-385; Maddon et al., 1986, Cell 47:333-348).
HIV infection is pandemic and HIV-associated diseases have become a world-wide health problem. Despite considerable efforts in the design of anti-HIV modalities, there is, thus far, no successful prophylactic or therapeutic regimen against AIDS. However, several stages of the HIV life cycle have been considered as potential targets for therapeutic intervention (Mitsuya et al., 1991, FASEB J. 5:2369-2381). For example, virally-encoded reverse transcriptase has been a major focus of drug development. A number of reverse-transcriptase-targeted drugs, including 2N,3N-dideoxynucleotide analogs such as AZT, ddI, ddC, and ddT have been shown to be active against HIV (Mitsuya et al., 1990, Science 249:1533-1544). While beneficial, these nucleotide analogs are not curative, probably due to the rapid appearance of drug resistant HIV mutants (Lander et al., 1989, Science 243:1731-1734). In addition, these drugs often exhibit toxic side effects, such as bone marrow suppression, vomiting, and liver abnormalities.
Another stage of the HIV life cycle that has been targeted is viral entry into cells, the earliest stage of HIV infection. This approach has primarily utilized recombinant soluble CD4 protein to inhibit infection of CD4+ T cells by some HIV-1 strains (Smith et al., 1987, Science 238:1704-1707). Certain primary HIV-1 isolates, however, are relatively less sensitive to inhibition by recombinant CD4 (Daar et al., 1990, Proc. Natl. Acad. Sci. USA 87:6574-6579). To date, clinical trials of recombinant, soluble CD4 have produced inconclusive results (Schooley et al., 1990, Ann. Int. Med. 112:247-253; Kahn et al., 1990, Ann. Int. Med. 112:254-261; Yarchoan et al., 1989, Proc. Vth Int. Conf. on AIDS, p. 564, MCP 137).
Additionally, the later stages of HIV replication (which involve crucial virus-specific processing of certain viral proteins and enzymes) have been targeted for anti-HIV drug development. Late-stage processing is dependent on the activity of a virally-encoded protease, and drugs including saquinavir, ritonavir, and indinavir have been developed to inhibit this protease (Pettit et al., 1993, Persp. Drug Discov. Design 1:69-83). With this class of drugs, the emergence of drug resistant HIV mutants is also a problem; resistance to one inhibitor often confers cross-resistance to other protease inhibitors (Condra et al., 1995, Nature 374:569-571). Also, these drugs often exhibit toxic side-effects such as nausea, altered taste, circumoral parethesias, fat deposits, diarrhea and nephrolithiasis.
Antiviral therapy of HIV using different combinations of nucleoside analogs and protease inhibitors have recently been shown to be more effective than the use of a single drug alone (Torres et al., 1997, Infec. Med. 14:142-160). However, despite the ability to achieve significant decreases in viral burden, there is no evidence to date that combinations of available drugs will afford a curative treatment for AIDS.
Other potential approaches for developing treatment for AIDS include the delivery of exogenous genes into infected cells. One such gene therapy approach involves the use of genetically-engineered viral vectors to introduce toxic gene products to kill HIV-infected cells. Another form of gene therapy is designed to protect virally-infected cells from cytolysis by specifically disrupting viral replication. Stable expression of RNA-based (decoys, antisense and ribozymes) or protein-based (transdominant mutants) HIV-1 antiviral agents can inhibit certain stages of the viral life cycle. A number of anti-HIV suppressors have been reported, such as decoy RNA of TAR or RRE (Sullenger et al., 1990, Cell 63:601-608; Sullenger et al., 1991, J. Virol. 65:6811-6816; Lisziewicz et al., 1993, New Biol. 3:82-89; Lee et al., 1994, J. Virol. 68:8254-8264), ribozymes (Sarver et al., 1990, Science 247:1222-1225; Wecrasinghe et al., 1991, J. Virol. 65:5531-5534; Dropulic et al., 1992, J. Virol. 66:1432-1441; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA 89:10802-10806; Yu et al., 1993, Proc. Natl. Acad. Sci. USA 90:6340-6344; Yu et al., 1995, Proc. Natl. Acad. Sci. USA 92:699-703; Yamada et al., 1994, Gene Therapy 1:38-45), antisense RNA complementary to the mRNA of viral gag, tat, rev or env genes (Sezakiel et al., 1991, J. Virol. 65:468-472; Chatterjee et al., 1992, Science 258:1485-1488; Rhodes et al., 1990, J. gen. Virol. 71:1965. Rhodes et al., 1991, AIDS 5:145-151; Sezakiel et al., 1992, J. Virol. 66:5576-5581; Joshi et al., 1991, J. Virol. 65:5524-5530) and transdominant mutants including Rev (Bevec et al., 1992, Proc. Natl. Acad. Sci. USA 89:9870-9874), Tat (Pearson et al., 1990, Proc. Natl. Acad. Sci. USA 87:5079-5083; Modesti et al., 1991, New Biol. 3:759-768), Gag (Trono et al., 1989, Cell 59:113-120), Env (Bushschacher et al., 1995, J. Virol. 69:1344-1348) and protease (Junker et al., 1996, J. Virol. 70:7765-7772).
Antisense polynucleotides have been designed to complex with and sequester the HIV-1 transcripts (Holmes et al., WO 93/11230; Lipps et al., WO 94/10302; Kretschmer et al., EP 594,881; and Chatterjee et al., 1992, Science 258:1485). Furthermore, an enzymatically active RNA, termed ribozyme, has been used to cleave viral transcripts. The use of a ribozyme to generate resistance to HIV-1 in a hematopoietic cell line has been reported (Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA 89:10802-06; Yamada et al., 1994, Gene Therapy 1:38-45; Ho et al., WO 94/26877; and Cech and Sullenger, WO 95/13379). In preclinical studies, RevM10, a transdominant Rev protein, has been transfected ex vivo into CD4xe2x88x92 cells of HIV-infected individuals and shown to confer survival advantage over cells transfected with vector only (Woffendin et al., 1996, Proc. Natl. Acad. Sci. USA 93:2889-2894).
Despite enormous efforts in the art, reliable, curative anti-HIV therapeutic agents and regimens have not been developed.
In nature, evolution of an intracellular pathogen such as HIV requires the development of interactions of its genes and gene products with multiple cellular components. For instance, the interactions of a virus with a host cell involves binding of the virus to a specific cellular receptor(s), translocation through the cellular membrane, uncoating, replication of the viral genome, transcription of the viral genes, etc. Each of these events occurs in a cell and involves interactions with a cellular component. Thus, the life cycle of a virus can be completed only if the cell is xe2x80x9cpermissivexe2x80x9d for viral infection. Availability of amino acids and nucleotides for replication of the viral genome and protein synthesis, energy status of the cell, the presence of cellular transcription factors and enzymes all contribute to the propagation of the virus in the cell. Consequently, the cellular components, in part, determine host cell susceptibility to infection, and can be used as potential targets for the development of new therapeutic interventions. In the case of HIV, one cellular component that has been used towards this end is the cell surface molecule for HIV attachment, CD4.
Recently, it was reported that HIV entry into a susceptible cell requires the expression of a second type of receptor, the chemokine receptors (CCR2, CCR3, CCR5 or CXCR4), in addition to CD4 (Moore, 1997, Science 276:51-52). A chemokine receptor normally binds RANTES, MIP-1xcex1 and MIP-1xcex2 as its natural ligand. In the case of HIV infection, it has been proposed that CD4 first binds to the HIV gp120 protein on the cell surface followed by binding of this complex to a chemokine receptor, resulting in viral entry into the cells (Cohen, 1997, Science 275:1261). Therefore, chemokine receptors can present an additional cellular target for the design of HIV therapeutic agents. Inhibitors of HIV/chemokine receptor interactions are being tested as anti-HIV agents. However, there remains a need for the discovery of additional cellular targets for the design of anti-HIV therapeutics, particularly intracellular targets for disrupting viral replication after viral entry into a cell.
It also has been reported that overexpression of the hdm2 protein drives oncogenesis through antagonism of the p53 tumor suppressor protein. Binding of hdm2 to p53 promotes the degradation of p53. Several recent reports focus on the requirement for the hdm2 protein to actively shuttle between nucleus and cytoplasm in order to exert inhibition of p53 (Lain et al., 1999, Exp. Cell Res. 248:457-472). An inhibitor of nuclear export activates the p53 response and induces the localization of HDM2 and p53 to U1A-positive nuclear bodies associated with the PODs (Roth et al., 1998, EMBO J. 15:554-564). Nucleo-cytoplasmic shuttling of the hdm2 oncoprotein regulates the levels of the p53 protein via a pathway also used by the human immunodeficiency virus rev protein (Tao et al., 1999, Proc. Natl. Acad. Sci. USA 96:3077-3080). P19(ARF) stabilizes p53 by blocking nucleo-cytoplasmic shuttling of Mdm2 (Tao et al., 1999, Proc. Natl. Acad. Sci. USA 96:6937-6941). Thus, shuttling of the hdm2 protein apparently depends on a nuclear export pathway that overlaps, or is identical to, that utilized by the HIV rev protein. Furthermore, the P19 (ARF) tumor suppressor stabilizes p53, thereby inhibiting tumorigenesis, by interfering with this nucleo-cytoplasmic shuttling.
Thus, there remains a need to isolate and identify genetic suppressor elements and cellular target genes that are involved in the inhibition of HIV infection or tumorigenesis.
The present invention relates to compositions and methods for inhibiting HIV infection by down-regulating expression of certain human cellular genes and/or inhibiting the activity of products encoded by such genes. In particular, it relates to a number of human cell-derived nucleic acid molecules which inhibit HIV infection in susceptible cells. The isolated nucleic acid molecules correspond to portions of cellular genes or complements thereof, and are referred to herein as genetic suppressor elements (GSEs). The cellular genes encode intracellular products necessary for productive HIV infection. Additionally, small molecule inhibitors of the same cellular genes and their encoded products are also within the scope of the present invention. The invention also relates to methods for identifying additional cellular genes as therapeutic targets for suppressing HIV infection, and methods for using such cellular genes and their encoded products for selecting additional inhibitors of HIV.
The invention is based, in part, on the Applicants"" discovery that nucleic acid molecules isolated from human cells can prevent both the activation of latent HIV-1 in a CD4+ cell line and productive HIV infection in such cells, and that such nucleic acid molecules correspond to fragments of certain human cellular genes. In that regard, any cellular or viral marker associated with HIV infection can be used to select for such nucleic acid molecules. An example of such a marker is CD4, which is conveniently monitored by using a specific antibody.
Based on substantial sequence identity (90%-100%), a number of the isolated GSEs correspond to portions of human cellular genes that encode different subunits of a mitochondrial enzyme complex, NADH dehydrogenase. In addition, inhibitors of this enzyme also inhibit HIV infection in susceptible host cells, including freshly isolated human CD4+ T cells. Furthermore, additional GSEs have been selected that have substantial sequence identity (90%-100%) with the following cell-derived nucleic acid molecules: 2-oxoglutarate dehydrogenase, M2-type pyruvate kinase/cytosolic thyroid hormone binding protein, calnexin, ADP-ribosylation factor 3, eukaryotic initiation factor 3, protein tyrosine phosphatase, herpesvirus-associated ubiquitin-specific protease, eukaryotic initiation factor 4B, CD44, phosphatidyl-inositol 3 kinase, elongation factor 1 alpha, bone morphogenic protein-1, double-strand break DNA repair gene protein, rat guanine nucleotide releasing protein, anti-proliferative factor (BTG-1), lymphocyte-specific protein 1, protein phosphatase 2A, squalene synthetase, eukaryotic release factor 1, GTP binding protein, importin beta subunit, cell adhesion molecule L1, U-snRNP associated cyclophilin, recepin, Arg/Abl interacting protein (ArgBP2A), keratin-related protein, p18 protein, p40 protein, glucosidase II, alpha enolase, macrophage inflammatory protein 1 alpha, tumor protein translationally-controlled 1 (TCTP1), BBC1, Nef interacting protein, Na+-D-glucose cotransport regulator gene protein, hsp90 chaperone protein, FK506-binding protein A1, Rox, beta signal sequence receptor, tumorous imaginal disc protein, cell surface heparin binding protein, SH2-containing inositol 5-phosphatase (referred to herein as SHIP), guanine nucleotide binding protein beta polypeptide 2-like 1 (referred to herein as GNB2L1), arginyl tRNA synthetase (referred to herein as ArgRS), ABC transporter, cell division cycle 42 GTP-binding protein (referred to herein as CDC42), cyclosporin-A 19 (referred to herein as Csa-19), src kinase p59 (referred to herein as FYN), cathepsin B (referred to herein as CTSB), cathepsin L and glutaredoxin (referred to herein as GLRX).
Among the GSEs selected to inhibit HIV infection, several function in the sense orientation, while others function in the antisense orientation. Not intending to be bound by any particular theory, the GSEs of the invention are believed to down-regulate a cellular gene by different mechanisms. The GSEs are expressed in a host cell by encoding RNA molecules that do or do not encode protein products. GSEs in the sense orientation can exert their effects as transdominant mutants or RNA decoys. Transdominant mutants are expressed proteins or peptides that competitively inhibit the normal function of a wild-type protein in a dominant fashion. RNA decoys are protein binding sites that titrate out these proteins. GSEs in the antisense orientation can exert their effects as antisense RNA; i.e. nucleic acid molecules complementary to the mRNA of the target gene. These nucleic acid molecules bind to mRNA and block the translation of the mRNA. Some antisense nucleic acid molecules can act directly at the DNA level to inhibit transcription. The down-regulation of a cellular gene by a GSE, in turn, removes a cellular component necessary for, for example HIV replication, resulting in an inhibition of HIV infection.
A wide range of uses are encompassed by the invention including, but not limited to, HIV treatment and prevention by transferring protective GSE compounds as inhibitory compositions into HIV-susceptible cell types. For example, GSEs can be transferred into T cells, particularly CD4+ T cells that are the major cell population targeted by HIV. Alternatively, GSEs can be transferred into hematopoietic stem cells in vitro followed by their engraftment in an autologous or histocompatible or even histoincompatible recipient. In another embodiment, any cells susceptible to HIV infection can be directly transduced or transfected with GSEs in vivo. In yet another embodiment, inhibitors of NADH dehydrogenase, 2-oxoglutarate dehydrogenase, M2-type pyruvate kinase/cytosolic thyroid hormone binding protein, calnexin, ADP-ribosylation factor 3, eukaryotic initiation factor 3, protein tyrosine phosphatase, herpesvirus-associated ubiquitin-specific protease, eukaryotic initiation factor 4B, CD44, phosphatidyl-inositol 3 kinase, elongation factor 1 alpha, bone morphogenic protein-1, double-strand break DNA repair gene protein, rat guanine nucleotide releasing protein, anti-proliferative factor (BTG-1), lymphocyte-specific protein 1, protein phosphatase 2A, squalene synthetase, eukaryotic release factor 1, GTP binding protein, importin beta subunit, cell adhesion molecule L1, U-snRNP associated cyclophilin, recepin, Arg/Abl interacting protein (ArgBP2A), keratin related protein, p18 protein, p40 protein, glucosidase II, alpha enolase, macrophage inflammatory protein 1 alpha, tumor protein translationally-controlled 1 (TCTP1), BBC1, Nef interacting protein, Na+-D-glucose cotransport regulator gene protein, hsp90 chaperone protein, FK506-binding protein A1, Rox, beta signal sequence receptor, tumorous imaginal disc protein, cell surface heparin binding protein, SH2-containing inositol 5-phosphatase (SHIP), guanine nucleotide binding protein beta polypeptide 2-like 1 (GNB2L1), arginyl tRNA synthetase (ArgRS), ABC transporter, cell division cycle 42 GTP-binding protein (CDC42), cyclosporin-A 19 (Csa-19), src kinase p59 (FYN), cathepsin B (CTSB), cathepsin L and glutaredoxin (GLRX) can be used as inhibitory compositions in vivo to suppress or prevent HIV infection.